IS LAB: WEEK 3 DILUTION AND ANTIGEN-ANTIBODY REACTION

DILUTION

To make weaker solutions from stronger solutions. Indication of relative concentration.

DILUTION FACTOR

Used to correct concentration after using a diluted sample. The result using the dilution must be multiplied by the reciprocal of the dilution made.

SINGLE DILUTION

Used when concentration of sample is too great to be determined accurately or there is low sample size available.

SERIAL DILUTION

For obtaining concentrations of solutions, multiply the original concentration by the first dilution, then by the second and third until the desired concentration is known.

ZONE OF EQUIVALENCE

Occurs when the number of multivalent sites of antigen and antibody are approximately equal.

FALSE-NEGATIVE REACTIONS

Failure to achieve the zone of equivalence may be cause by ___________

PROZONE PHENOMENON

ANTIBODY EXCESS

POSTZONE PHENOMENON

ANTIGEN EXCESS

SERUM DILUTION

REMEDY FOR PROZONE PHENOMENON

REPEAT TESTING AFTER 1-2 WEEKS

REMEDY FOR POSTZONE PHENOMENON

PRECIPITATION

Binding of antibodies + soluble antigens resulting to insoluble complexes

AGGLUTINATION

Binding of antibodies + particulate antigens forming cellular aggregates

PRECIPITATION REACTIONS

PRECIPITATION IN A FLUID MEDIUM

PRECIPITATION BY PASSIVE IMMUNODIFFUSION

PRECIPITATION BY ELECTROPHORETIC TECHNIQUES

NEPHELOMETRY

More sensitive than turbidimetry. Can be used to detect either antigen or antibody.

NEPHELOMETRY

Measures the amount of light that is scattered at a particular angle from the incident beam as it passes through a suspension.

HIGHER

The lower the light intensity, the _______ the concentration of complexes

TURBIDIMETRY

A detection device is placed in direct line with an incident light, measures the reduction in light intensity caused by reflection, absorption or scatter.

PRECIPITATION IN A FLUID MEDIUM

TURBIDIMETRY

NEPHELOMETRY

PRECIPITATION BY PASSIVE IMMUNODIFFUSION

OUDIN’S TECHNIQUE - SINGLE DIFFUSION

OAKLEY-FULTHORPE TECHNIQUE - DOUBLE DIFFUSION

OUDIN’S TECHNIQUE

Only antigen is diffusing

OAKLEY-FULTHORPE TECHNIQUE

Intervening column plain agar is placed in between. Both antigen and antibody diffusing.

OUDIN’S TECHNIQUE

OAKLEY-FULTHORPE TECHNIQUE

SINGLE DIFFUSION IN TWO DIMENSIONS

Single radial immunodiffusion (SRID). Uses a flat surface petri dish or slide for precipitation reaction. Petri dish allows radial diffusion.

PRECIPITIN RING

SINGLE DIFFUSION IN TWO DIMENSIONS (+) RESULTS

MANCINI METHOD

ENDPOINT METHOD

FAHEY-MCKELVEY METHOD

KINETIC METHOD

MANCINI METHOD

Antigen is allowed to diffuse completely.

ANTIGEN CONCENTRATION

MANCINI METHOD: DIAMETER SQUARED = _________

LOG OF AG CONCENTRATION

FAHEY-MCKELVEY METHOD: INTERPRETATION: d =________

FAHEY-MCKELVEY METHOD

Antigen is NOT allowed to diffuse completely

FAHEY-MCKELVEY METHOD

Measurements taken before the point of equivalence is reached. 18 hours of incubation.

QUALITATIVE OUCHTERLONY TECHNIQUE

Uses a flat surface petri dish or slide for precipitation reaction. (+) Result: Distinct pattern of precipitation

QUALITATIVE OUCHTERLONY TECHNIQUE

IDENTICAL

SMOOTH CURVE

NON-IDENTICAL

CROSSED LINES

PARTIALLY IDENTICAL

SPUR. THE SPUR IS POINTING TO THE SIMPLER ANTIGEN

IDENTICAL, SMOOTH CURVE

NON-IDENTICAL, CROSSED LINES

PARTIALLY IDENTICAL, SPUR

IMMUNOELECTROPHORESIS

Antigen: usually from the patient serum

Antibody: placed on trough cut parallel to separated proteins

Gel is incubated for 18 to 24 hours.

MONOCLONAL GAMMOPATHY

IMMUNOELECTROPHORESIS ABNORMAL COUNTOUR OF PRECIPITIN ARC MAY INDICATE ____________

IMMUNOFIXATION ELECTROPHORESIS

Used to identify heavy and light chains involved in monoclonal gammopathies

HEAVY CHAINS

gamma, alpha, mu

LIGHT CHAINS

Kappa, lambda

SENSITIZATION

Initial reaction which involves Ag-Ab combination through single antigenic determinants on the particle surface.

SENSITIZATION

Initial reaction follows the law of mass action is rapid and reversible.

LATTICE FORMATION

Formation of cross-links that form the visible aggregates, represents the stabilization of Ag-Ab complexes with the binding together of multiple antigenic determinants.

DIRECT AGGLUTINATION

Antigen are found naturally on particle. Example: ABO blood typing, widal’s test for salmonella.

TYPE O

INDIRECT AGGLUTINATION

Employs particles that are coated with antigens not normally found on their surfaces. Example: Latex agglutination test.

REVERSE PASSIVE AGGLUTINATION

Antibodies rather than antigens are attached to a carrier particle. DETECTS ANTIGENS. EXAMPLE: CRP

COAGGLUTINATION

Uses bacterial protein as inert particles to which antibodies are attached. DETECTS ANTIGENS. EXAMPLE: PROTEIN A OF S.AUREUS

AGGLUTINATION INHIBITION

Based on competition between particulate and soluble antigens for limited antibody-combining sites.

LACK OF AGGLUTINATION

AGGLUTINATION INHIBITION: (+) RESULTS:

HEMAGGLUTINATION INHIBITION AND RESPIRATORY SYNCYTIAL VIRUS

EXAMPLE OF AGGLUTINATION INHIBITION:

ANTI-HUMAN GLOBULIN MEDIATED AGGLUTINATION

Employs an antihuman globulin to detect antibody-coated cells.

COOMB’S TEST

AHG TEST

ANTI-HUMAN GLOBULIN MEDIATED AGGLUTINATION EXAMPLES:

24 HRS

IgG

50-72 HRS

IgM

ANTIGEN CONCENTRATION

D2 =